Glycosides of catechol estrogens

ABSTRACT

There are described novel glycosides of catechol estrogen, a method of preparing the same, and a medicament comprising one of the glycosides as an active ingredient. The glycosides are shown by the formula of ##STR1## wherein X is carbonyl group or ##STR2##  R 10  is hydroxyl group or glycosyloxy group, and R 2  is a hydrogen atom or ethynyl group; R 11  is a hydrogen atom, hydroxyl group, or glycosyloxy group; R 12  is hydroxyl group or glycosyloxy group; and R 13  is hydroxyl group or glycosyloxy group, 
     in which glycosyloxy group is selected from the group consisting of glucosyloxy, galactosyloxy, mannosyloxy, arabinosyloxy, ribosyloxy, xylosyloxy, fructosyloxy, rhamnosyloxy, fucosyloxy, maltosyloxy, cellobiosyloxy, lactosyloxy, sucrosyloxy, maltotriosyloxy, maltotetraosyloxy, maltopentaosyloxy, maltohexaosyloxy, maltoheptaosyloxy, and sialosyloxy, and in this case, at least one of R 10 , R 11 , R 12 , and R 13  is glycosyloxy group as defined above.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel glycosides of catechol estrogens,a process for the manufacture thereof, and a medicament comprising atleast one of the glycosides for the prevention and treatment of lipidperoxide- and free radical-related diseases.

2. Related Arts

It is known that lipid peroxides are causative of ischemia-reperfusioninjury, ischemic heart disease, atherosclerosis, retinopathy in infant,siderosis, cataract, hepatitis, pancreatitis, diabetic microangiopathy,melanoderma, liver spots, pre-eclampsia and aging itself. Therefore, theprevention of lipid peroxidation by use of suitable antioxidants isbeneficial for the prophylactic and treatment of such diseases. Thus, itis in need to develop effective antioxidants as medical compound. In1987, Yoshino et al. reported that estrogens and their metabolitescatechol estrogens are effective to prevent an increase in lipidperoxide levels in mice [K. Yoshino et al., "J. Clin. Biochem. Nutr.",Vol. 3, pages 233-240 (1987)].

Thus, estrogens and catechol estrogens were thought to be hopeful forclinical application, however, the following disadvantages thereof makeit difficult: Since these compounds are scarcely soluble in water, it isdifficult to prepare an injection. Estrogens are not applicable to menbecause of their activity as a female hormone. Although estrogenicactivity of catechol estrogens is almost nil, they are unstable againstexposure to air and light so that the development of a medicamentcomprising catechol estrogen is not progressing. In addition, it isknown that a half life of catechol estrogens in human blood is veryshort due to high susceptibility to be taken up into red blood cells andto be metabolized by catechol-O-methyl-transferase in the cells.Accordingly, these characteristics of catechol estrogens make itdifficult to develop useful medicaments, even though they have a potentantioxidant activity.

As to organic synthesis of catechol estrogens, not a few papers werereported in literatures as listed below:

(a) J. Fishman, "J. Am. Chem. Soc.", 80, 1213 (1958),

(b) P. N. Rao, et al., "Tetrahedron", 10, 144 (1960),

(c) J. Fishman, "J. Org. Chem.", 25, 585 (1960),

(d) T. Nambara, et al., "Chem. Pharm. Bull.", 18, 474 (1970),

(e) T. Nambara, et al., "Chem. Pharm. Bull.", 18, 1191 (1970),

(f) I. Yoshizawa, et al., "Chem. Pharm. Bull.", 20, 1842 (1972),

(g) K. Kovacs, et al., "Acta Phys. Chem.", 19, 287 (1973),

(h) H. G. Gelbke, et al., "Steroids", 21, 205 (1973),

(i) G. Stubenrauch, et al., "Steroids", 28, 733 (1976),

(j) R. G. Xie, et al., "Steroids", 40, 389 (1982), and

(k) T. Ohkubo, et al., "Chem. Pharm. Bull.", 36, 419 (1988),

Among the methods previously available, only two methods gave arelatively good yield of 50 to 60% for the preparation of catecholestrogens. Namely, the method described in Reference (j) includes theFriedel-Crafts reaction of estradiol to form 2-acetylestradiol17-acetate (yield: 57.3%) followed by the Dakin reaction at pH 8.2-8.5to form 2-hydroxyestradiol 17-acetate (yield: 85.2%). In this case, theoverall yield was 49%. According to the method described in Reference(f), estradiol was reacted with benzoyl peroxide to form2-hydroxyestradiol 2-monobenzoate and followed by hydrolysis to give2-hydroxyestradiol. The overall yield in this process was 60%.

When derivatives of catechol estrogens are synthesized, it is importantto prepare desirable intermediates thereof, wherein a suitableprotective group is introduced into desired position. For example,Nambara et al. [References (d) and (e)] described the preparation of2-acetoxyestrogens 3-alkyl ether and3-acetoxy-2-alkoxy-1,3,5(10)-estratriene compounds from correspondingacetyl estrogens by Baeyer-Villiger oxidation in yields of 65-75% and56-60%, respectively, in order to introduce desired substitution intodesired positions.

For the preparation of 2-hydroxyestrogen 3-glycoside,2-benzyloxyestrogen is an important intermediate. The proceduredescribed in References (d) and (e) involved 11 reaction steps for thesynthesis of 2-benzyloxyestradiol 17-acetate from 3-deoxyestradiolacetate and its overall yield was only 6%, then, 2-benzyloxyestradiol17-acetate was further subjected to glucuronidation to form2-hydroxyestradiol 3-glucuronide in a overall yield of 0.33%.

Thus, the procedures previously reported are not suitable for themanufacture thereof, because starting material is expensive, the processinvolves many reaction steps, and yield is not satisfactory.

SUMMARY OF THE INVENTION

An object of the invention is to provide novel glycosides of catecholestrogens which have an effective antioxidant activity, high solubilityin water, and sufficient stability in body fluid and during the processof manufacture thereof.

Another object of the invention is to provide suitable processes for themanufacture of catechol estrogen compounds, by taking the followingpoints into consideration:

(a) The starting material is available at reasonable cost;

(b) The number of reaction steps is minimum;

(c) Reaction time is as short as possible, and

(d) Yield is satisfactory.

The present inventors energetically studied and investigated to find asimple process for preparing 2-acetylestrogens with a good yield, fromwhich they successfully prepared various glycosylated compounds ofcatechol estrogens. The glycosylated compounds thus obtained were met totheir needs mentioned above.

The process according to the invention is shown in the following Scheme.##STR3##

In the Scheme, Bn is benzyl group; X is carbonyl group or ##STR4## Y iscarbonyl group or ##STR5## Z is carbonyl group or ##STR6## R₁ isacetoxyl group; R₂ is a hydrogen atom, or ethynyl group; R₃ is ahydrogen atom or acetoxyl group; R₄ is a protective group with an acetaltype; R₆ is glycosyl group whose functional groups are protected; R₈ ishydroxyl group; R₉ is a hydrogen atom or hydroxyl group; R₁₀ is hydroxylgroup or glycosyloxy group; R₁₁ is a hydrogen atom, hydroxyl group orglycosyloxy group; however R₁₀ and/or R₁₁ is glycosyloxy group; R_(12')is glycosyloxy group; and R_(13') is glycosyloxy group; whereinglycosyloxy group is selected from the group consisting of glucosyloxy,galactosyloxy, mannosyloxy, arabinosyloxy, ribosyloxy, xylosyloxy,fructosyloxy, rhamnosyloxy, fucosyloxy, maltosyloxy, cellobiosyloxy,lactosyloxy, sucrosyloxy, maltotriosyloxy, maltotetraosyloxy,maltopentaosyloxy, maltohexaosyloxy, maltoheptaosyloxy, and sialosyloxy.

As a result, it was found that 2-acetylestrogens of the formula:##STR7## wherein Y and R₃ have the meanings as defined above, are usefulas intermediates for the synthesis of catechol estrogens and glycosidesthereof to open the way for the present invention.

The following section describes the method of the invention in detail.2-Acetylestrogens, which are key intermediates in the present invention,can be synthesized by subjecting estrogen 3-acetate to Friesrearrangement reaction. The important point in this process is that thereaction is carried out in the presence of acetyl chloride. It was foundthat the yield of 2-acetylestrogen from the starting material wasincreased to over 80% in the presence of acetyl chloride. The preferableamount of acetyl chloride is in a range of 0.1 to 0.6 in terms of molarratio to estrogen 3-acetate.

The synthesis of 16- and/or 17-glycosylated catechol estrogens iscarried out via catechol estrogens that is prepared from2-acetylestrogens by Dakin reaction. The previously reported Dakinreaction was carried out at pH below 9, so that the reaction took longperiod of time. On the contrary, the present invention found thatreaction time could be lessened and yield of the product was increasedto over 90% when Dakin reaction was carried out at pH 11-13.

2-Acetylestrogens are subjected to Baeyer-Villiger oxidation reaction toobtain 2-acetoxyestrogens. The previously available Baeyer-Villigeroxidation was carried out in the absence of an alkali metal salt,requiring long time to complete the reaction. On the contrary, thepresent invention found that Baeyer-Villiger oxidation in the presenceof the alkali metal salt required only 1 or 2 days to complete thereaction by >80% yield. Alkali metal salt available in the presentinvention is selected from the followings; disodium hydrogen phosphate,dipotassium hydrogen phosphate, sodium hydrogen carbonate, and potassiumhydrogen carbonate. Any amount of the alkali metal salt is useful andits preferable amount is in a range between 0.1 and 5 in terms of molarratio to 2-acetylestrogens. As to protective groups for hydroxyl groupat C-3 position during Baeyer-Villiger oxidation, any radical can beused such as methyl, benzyl, t-butyldimethylsilyl, triisopropylsilyl,acetyl, benzoyl, methoxymethyl, tetrahydropyranyl, and so on.

A process for preparing 3-glycosides of catechol estrogens involves2-benzyloxyestrogens as an intermediate compound. It is prerequisitethat the protection of hydroxyl group at C-3 must be stable duringBaeyer-Villiger oxidation, hydrolysis of acetyl group, and benzylation.It was found that protective groups having an acetal type such asmethoxymethyl, 2-methoxyethoxymethyl, and tetrahydropyranyl groups aresatisfactory in this point of view. 2-Benzyloxyestrogens can besynthesized as follows: The hydroxyl group at C-3 of 2-acetylestrogensis protected by an acetal group to form 2-acetylestrogen 3-acetalcompounds, subjecting to Baeyer-Villiger oxidation to form2-acetoxyestrogen 3-acetal compounds, subjecting to hydrolysis to form2-hydroxyestrogen 3-acetal compounds, subjecting to benzylation to form2-benzyloxyestrogen 3-acetal compounds, and followed by elimination of3-acetal group. According to the method of this invention,2-benzyloxyestrogens can be synthesized from estrogens by 70% yield.

This invention also provides a short process for the preparation of3-glycosides of catechol estrogens. That is, 2-acetylestrogen isdirectly glycosylated, 2-acetylestrogen 3-glycosides thus obtained aresubjected to Baeyer-Villiger oxidation to form 2-acetoxyestrogen3-glycosides, followed by hydrolysis of protective groups. According tothis invention, both 2-hydroxyestrogen 3-(α-glycoside) and2-hydroxyestrogen 3-(β-glycoside) were synthesized from estrogens by 30%yield for each.

The following section describes the process for the manufacture ofcatechol estrogen glycosides from estradiol as starting material. Thefirst step is that estradiol was reacted with acetic anhydride inpyridine at room temperature for overnight, yielding estradiol diacetatestoichiometrically. Estradiol diacetate was heated at 80° C. innitrobenzene for 1 hour in tile presence of 2 mol equivalent of aluminumchloride and 0.4 mol equivalent of acetyl chloride, and then2-acetylestradiol 17-acetate was obtained by 85% yield.2-Acetylestradiol 17-acetate was reacted with hydrogen peroxide at roomtemperature for 2 hours maintaining pH in a range between 12.0 and 12.5by the addition of sodium hydroxide and 2-hydroxyestradiol 17-acetatethus obtained was hydrolyzed by conventional method either in acidic oralkaline condition to form 2-hydroxyestradiol by overall yield of 77%.

The catechol moiety of 2-hydroxyestradiol was protected by benzyl groupand the protected compound was reacted with acetobromoglucose in thepresence of silver carbonate, followed by elimination of protectivegroups and desired 2-hydroxyestradiol 17-glucoside was obtained.

2-Acetylestradiol 17-acetate was reacted with chloromethyl methyl etherin tetrahydrofuran containing sodium hydrogen, yielding2-acetylestradiol 17-acetate 3-methoxymethyl ether stoichiometrically,subjecting to Baeyer-Villiger oxidation with m-chloroperbenzoic acid inchloroform containing disodium hydrogen phosphate, and thus2-acetoxyestradiol 17-acetate 3-methoxymethyl ether was formed by 88%yield.

As to the use of 2-acetoxyestradiol 17-acetate 3-methoxymethyl ether ina process of the 2-benzyloxyestradiol 17-acetate production, a fewprocedures can be useful. First, 2-acetatoxyestradiol 17-acetate3-methoxymethyl ether was converted to 2-hydroxyestradiol 17-acetate3-methoxymethyl ether by the reaction with potassium hydrogen carbonatein a mixture of acetone-water-methanol. 2-Hydroxyestradiol 17-acetate3-methoxymethyl ether was reacted with benzyl chloride in ethanol in thepresence of potassium carbonate, yielding a mixture of 17-acetoxy- and17-hydroxy-2-benzyloxy compounds. The mixture of 2-benzyloxy compoundswas further treated with acetic anhydride-pyridine. Whenphenyldiazomethane was used as a benzylating agent, acetyl group at C-17was retained. Methoxymethyl group of 2-benzyloxyestradiol 17-acetate3-methoxymethyl ether was eliminated to give 2-benzyloxyestradiol17-acetate. Second, 2-acetatoxyestradiol 17-acetate 3-methoxymethylether was hydrolyzed in methanol in the presence of sodium hydroxide,and 2-hydroxyestradiol 3-methoxymethyl ether thus obtained was reactedwith benzyl chloride in ethanol in the presence of potassium carbonateto form 2-benzyloxyestradiol 3-methoxymethyl ether. 17-Hydroxyl group of2-benzyloxyestradiol 3-methoxymethyl ether was reacted with aceticanhydride-pyridine for acetylation, followed by elimination ofmethoxymethyl group to give 2-benzyloxyestradiol 17-acetate. Third, theintermediate 2-benzyloxyestradiol 3-methoxymethyl ether was subjected tohydrolysis in methanol containing HCl to form 2-benzyloxyestradiol,followed by acetylation of hydroxyl group at C-3 and C-17 in a solutioncontaining acetic anhydride-pyridine. Then, 2-benzyloxyestradiol17-acetate was obtained by selective hydrolysis in a mixture ofacetone-water-methanol in the presence of potassium hydrogen carbonate.

These three different processes provide 2-benzyloxyestradiol 17-acetatestoichiometrically from 2-acetoxyestradiol 17-acetate 3-methoxymethylether. 2-Benzyloxyestradiol 17-acetate can be reacted withacetobromoglucose in the presence of CdCO₃ and after deprotection,2-hydroxyestradiol 3-glucoside was obtained.

When 2-acetylestradiol 17-acetate was reacted with acetobromoglucose intoluene containing CdCO₃, two anomers,2,3,4,6-tetra-O-acetyl-1-O-(2-acetyl-17β-acetoxy-1,3,5(10)-estratriene-3-yl)-β-D-glucopyranosideand2,3,4,6-tetra-O-acetyl-1-O-(2-acetyl-17β-acetoxy-1,3,5(10)-estratriene-3-yl)-α-D-glucopyranoside,were formed by 39% and 48% yield, respectively. Each anomer wassubjected to Baeyer-Villiger oxidation in the presence of an alkalimetal salt to form 2-acetoxyl compounds, followed by elimination ofacetyl group under alkaline condition to form 2-hydroxyestradiol3-(β-D-glucopyranoside) or 2-hydroxyestradiol 3-(α-D-glucopyranoside) by83% and 70% yield, respectively. Overall yield of β-anomer and α-anomerwas 28% and 29% respectively, from estradiol as starting material.

2-Acetylestradiol 17-acetate was transformed to 2-acetylestradiol17-acetate 3-benzyl ether, subjecting to Baeyer-Villiger oxidation inthe presence of an alkali metal salt to form 2-acetoxyestradiol17-acetate 3-benzyl ether, subjecting selective hydrolysis at C-2 toform 2-hydroxyestradiol 17-acetate 3-benzyl ether. 2-Hydroxyestradiol17-acetate 3-benzyl ether was reacted with acetobromoglucose in thepresence of CdCO₃, and after deprotection, 2-hydroxyestradiol2-glucoside was obtained.

The present inventors have found that novel glycosides of catecholestrogens acquired an increased hydrophilicity. For example, the volumeof water required to dissolve 1 mg of various glycosides is asfollowing; 2 to 3 ml for 17-glucoside, 2 ml for 17-maltoside, 0.7 ml for2-glucoside, and 3 ml for 3-glucoside, while 1 mg of 2-hydroxyestradiolcannot be dissolved in 3 ml of water. Accordingly, the glycosidesobtained in the present invention provide a way to prepare an injectioncontaining the same.

It was also found that glycosides of catechol estrogens retainedantioxidant activity in the same degree to that of parent catecholestrogens and that the rate of incorporation of the glycosides into redblood cells was extremely decreased, indicating that half life of theglycosides was much improved.

The glycosides of catechol estrogens obtained by the present inventionhave not estrogenic activity at the dosage required for antioxidantactivity. The glycosides of catechol estrogens provide medicaments withdesirable types such as injection, powder, tablets, capsules, ointment,and so on.

PREFERRED EMBODIMENTS OF THE INVENTION

The invention will now be further explained in more detail withreference to Manufacturing Examples, Test Examples and MedicinePreparation Examples.

EXAMPLE 1

a) Estradiol diacetate

A mixture of estradiol (30 g, 110 mmol), pyridine (300 ml, 3.71 mol) andacetic anhydride (105 ml, 1.08 mol) was stirred overnight at roomtemperature. The reaction mixture was poured onto ice (1500 g) andprecipitate was collected by filtration, washed with water, and dried invacuo to afford 39.3 g (100%) of the titled compound.

¹ H-NMR spectrum (DMSO-d₆) δ ppm: 0.78 (3H, s, 18-CH₃), 1.1-2.9 (15H, m,CH and CH₂), 2.01 (3H, s, C₁₇ --OCOCH₃), 2.23 (3H, s, C₃ --OCOCH₃), 4.62(1H, t like, C₁₇ --H), 6.83 (2H, m, C₂ --H and C₄ --H), and 7.28 (1H, d,J=10 Hz, C₁ --H).

b) 2-Acetylestradiol 17-acetate

To a solution of estradiol diacetate (25.0 g, 70.1 mmol) in nitrobenzene(250 ml), AlCl₃ (18.6 g, 139 mmol) and acetyl chloride (2.0 ml, 28 mmol)were added, and the solution was stirred for 1 hour at 80° C. Thereaction mixture was cooled and poured onto ice (1000 g). To thissolution concentrated HCl (100 ml) was added. After extraction withCHCl₃, the organic layer was washed successively with 1N-HCl, saturatedNaHCO₃ solution, and brine, and concentrated in vacuo to obtain a crudeproduct (25.3 g), to which methanol (MeOH) (150 ml) was added to afford17.7 g (70.6%) of the titled compound as an insoluble material. Thefiltrate was concentrated in vacuo and subjected to silica gelchromatography (elution solvent, CHCl₃ :hexane=2:1) to afford additional3.48 g of the titled compound. The total yield was 85%.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.84 (3H, s, 18-CH₃), 1.2-3.0 (15H, m,CH and CH₂), 2.07 (3H, s, C₁₇ --OCOCH₃), 2.59 (3H, s, C₂ --COCH₃), 4.70(1H, t like, C₁₇ --H), 6.67 (1H, s, C₄ --H), 7.59 (1H, s, C₁ --H), and12.03 (1H, s, C₃ --OH).

EXAMPLE 2 2-Hydroxyestradiol 17-acetate

To a mixture of 2-acetylestradiol 17-acetate (1.00 g, 2.81 mmol)obtained by the process described in Example 1-b, dioxane (30 ml), andwater (5 ml), 30% aqueous solution of hydrogen peroxide (0.600 ml, 5.28mmol) was added over 10 minutes and subsequently 2N-NaOH (2.3 ml, 4.6mmol) was added dropwise over 1.5 hours during which the pH of thesolution was maintained within a range of 11.0-12.5. After the reactionmixture was stirred for further 30 minutes, the pH of the solution wasbrought to 4.4 by addition of acetic acid (10 ml). To this solution Na₂SO₃ solution was added. After extraction with ethyl acetate (AcOEt), theorganic layer was washed with brine, dried over anhydrous Na₂ SO₄, andevaporated in vacuo to obtain a crude product (1.14 g), which was, inturn, subjected to silica gel chromatography (elution solvent,hexane:AcOEt=2:1) to afford 788 mg (85%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.82 (3H, s, 18-CH₃), 1.2-2.9 (15H, m,CH and CH₂), 2.08 (3H, s, C₁₇ --OCOCH₃), 4.75 (1H, t like, C₁₇ --H),5.44 (1H, s, Ar--OH), 5.97 (1H, s, Ar--OH), 6.59 (1H, s, C₄ --H), and6.80 (1H, s, C₁ --H).

Silica gel TLC: Rf=0.31 (hexane:AcOEt=2:1).

EXAMPLE 3

a) 2-Hydroxyestradiol 17-acetate

To a mixture of 2-acetylestradiol 17-acetate (80.5 g, 226 mmol) obtainedby the process described in Example 1-b, dioxane (2500 ml), and water(250 ml), 30% aqueous solution of hydrogen peroxide (40.3 ml, 355 mmol)was added and subsequently 2N-NaOH (193 ml, 386 mmol) was added dropwiseover 1.5 hours during which the pH of the solution was maintained withina range of 12.0-12.5. After the reaction mixture was stirred for further30 minutes, the pH of the solution was brought to 4.5 by addition ofacetic acid (800 ml). To this solution Na₂ SO₃ solution was added. Afterextraction with AcOEt, the organic layer was washed with brine, driedover anhydrous Na₂ SO₄, and evaporated in vacuo to obtain a crudeproduct (100 g) of the titled compound.

b) 2-Hydroxyestradiol

To the crude product (100 g) of 2-hydroxyestradiol 17-acetate obtainedby the process described in Example 3-a, MeOH (3600 ml) and concentratedHCl (400 ml) were added, and the solution was refluxed for 1 hour. Afterthe reaction mixture was cooled, water and AcOEt were added to it andthe solution was neutralized by addition of NaHCO₃, and extracted withAcOEt. The organic layer was washed with brine, and dried over anhydrousNa₂ SO₄, and then evaporated in vacuo to obtain a crude product (68.7g), which was, in turn, subjected to silica gel chromatography (elutionsolvent, hexane:AcOEt=1:1) to afford 59 g (91% from 2-acetylestradiol17-acetate) of the titled compound.

¹ H-NMR spectrum (DMSO-d₆) δ ppm: 0.66 (3H, s, 18-CH₃), 0.8-2.7 (15H, m,CH and CH₂), 3.52 (1H, m, C₁₇ --H), 4.48 (1H, d, J=5 Hz, C₁₇ --OH), 6.39(1H, s, C₄ --H), 6.62 (1H, s, C₁ --H), 8.42 (1H, s, Ar--OH), and 8.48(1H, s, Ar--OH).

Silica gel TLC: Rf=0.30 (hexane:AcOEt=1:1).

MS spectrum (EI/DI) m/z: 288 (M⁺).

EXAMPLE 4

a) 2-Hydroxyestradiol 2,3-dibenzyl ether

To a solution of 2-hydroxyestradiol 10.0 g (37.4 mmol) obtained by theprocess described in Example 3-b in ethanol (EtOH, 200 ml), K₂ CO₃ (12.0g) and benzyl chloride (10.0 g) were added, and the mixture was refluxedfor 5 hours. After the reaction mixture was cooled to room temperature,it was then filtered to remove an insoluble material. The insolublematerial was washed with AcOEt. The filtrate and washings were combinedand concentrated in vacuo to obtain a crude product, which was, in turn,subjected to silica gel chromatography (elution solvent,hexane:AcOEt=4:1) to afford 13.0 g (80.5%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.77 (3H, s, 18-CH₃), 1.0-2.9 (15H, m,CH and CH₂), 3.72 (1H, t like, C₁₇ --H), 5.11 (4H, s, C₆ H₅ CH₂ x 2),6.68 (1H, s, C₄ --H), 6.92 (1H, s, C₁ --H), and 7.2-7.5 (10H, m, C₆ H₅ x2).

Silica gel TLC: Rf=0.58 (hexane:AcOEt=1:1).

b)2,3,4,6-Tetra-O-acetyl-1-O-(2,3-dibenzyloxy-1,3,5(10)-estratriene-17β-yl)-β-D-glucopyranoside

Ag₂ CO₃ (471 mg, 1.71 mmol) was added to a solution of2-hydroxyestradiol 2,3-dibenzyl ether (200 mg, 0.427 mmol) obtained bythe process described in Example 4-a in benzene (20 ml), and thesuspension was distilled until 10 ml of benzene had been removed over 50minutes to remove moisture. A solution of acetobromoglucose (527 mg,1.28 mmol) in benzene (30 ml) was added dropwise to the stirred mixtureover 2 hours and an equal volume of benzene was simultaneously distilledto give a constant volume of the reaction mixture. Distillation wascontinued for further 1 hour, during which benzene was added dropwise tomaintain the volume of the suspension. An insoluble material was removedoff by filtration through a pad of Celite and washed with benzene andCH₂ Cl₂. The filtrate and washings were combined and evaporated in vacuoto obtain a crude product, which was, in turn, subjected to silica gelthin-layer chromatography (developing solvent, hexane:AcOEt=2.5:1) toafford 263 mg (77.1%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.74 (3H, s, 18-CH₃), 1.0-2.9 (15H, m,estrogen CH and CH₂), 2.01, 2.03, 2.07 and 2.09 (3H x 4, s x 4, OCOCH₃ x4), 3.63 (2H, m, C₁₇ --H and pyranose C₅ --H), 4.0-4.4 (2H, m, pyranoseC₆ --H₂), 4.56 (1H, d, J=8 Hz, pyranose C₁ --H), 4.8-5.3 (3H, m,pyranose C₂ --H, C₃ --H and C₄ --H), 5.11 (4H, s, C₆ H₅ CH₂ x 2), 6.67(1H, s, Ar C₄ --H), 6.89 (1H, s, Ar C₁ --H), and 7.1-7.6 (10H, m, C₆ H₅x 2) .

IR spectrum (KBr) cm⁻¹ : 2924, 1756, 1504, 1452, 1368, 1222, 1036, 736,and 698.

MS spectrum (EI/DI) m/z: 798 (M⁺).

Silica gel TLC: Rf=0.37 (hexane:AcOEt=2:1).

c)1-O-(2,3-Dibenzyloxy-1,3,5(10)-estratriene-17β-yl)-β-D-glucopyranoside

To2,3,4,6-tetra-O-acetyl-1-O-(2,3-dibenzyloxy-1,3,5(10)-estratriene-17β-yl)-β-D-glucopyranoside(1.04 g, 1.30 mmol) obtained by the process described in Example 4-b,MeOH (73 ml) was added. After the mixture was mildly heated to obtain ahomogeneous solution, 1N-NaOH (30 ml) was added, and the mixture wasstirred for 3 hours. After removal of MeOH by evaporation, the residuewas extracted with AcOEt. The organic layer was washed with brine, driedover anhydrous Na₂ SO₄, and then concentrated in vacuo to obtain a crudeproduct (865 mg), which was, in turn, subjected to silica gelchromatography (elution solvent, CHCl₃ :MeOH=10:1) to afford 613 mg(74.7%) of the titled compound.

¹ H-NMR spectrum (CD₃ OD) δ ppm: 0.83 (3H, s, 18-CH₃), 1.0-2.8 (15H, m,estrogen CH and CH₂), 3.1-4.0 (7H, m, C₁₇ --H and pyranose CH--O-- andCH₂ --O--), 4.35 (1H, d, J=8 Hz, pyranose C₁ --H), 5.01 (4H, s, C₆ H₅CH₂ x 2), 6.65 (1H, s, Ar C₄ --H), 6.86 (1H, s, Ar C₁ --H), and 7.2-7.4(10H, m, C₆ H₅ x 2).

IR spectrum (KBr) cm⁻¹ : 3420, 2920, 1502, 1452, 1262, 1074, 1014, 734,and 696.

MS spectrum (FAB, positive): 630 (M⁺).

Silica gel TLC: Rf=0.28 (CHCl₃ :MeOH=10:1).

d) 2-Hydroxyestradiol 17-(β-D-glucopyranoside)

To a solution of1-O-(2,3-dibenzyloxy-1,3,5(10)-estratriene-17β-yl)-β-D-glucopyranoside(1.89 g, 3.00 mmol) obtained by the process described in Example 4-c inMeOH (150 ml), a suspension of 5% Pd/C (200 mg) in MeOH (200 ml) wasadded, and the mixture was stirred under bubbling of H₂ for 20 hours.The catalyst Pd/C was removed off by filtration through a pad of Celite.The filtrate was evaporated in vacuo to obtain a crude product (1.39 g),which was, in turn, subjected to silica gel chromatography (elutionsolvent, CHCl₃ :MeOH=8:1) to afford 1.21 g (89.7%) of the titledcompound.

¹ H-NMR spectrum (CD₃ OD) δ ppm: 0.86 (3H, s, 18-CH₃), 1.0-2.8 (15H, m,estrogen CH and CH₂), 3.0-4.0 (7H, m, C₁₇ --H and pyranose CH--O-- andCH₂ --O--), 4.37 (1H, d, J=8 Hz, pyranose C₁ --H), 6.44 (1H, s, Ar C₄--H), and 6.69 (1H, s, Ar C₁ --H).

¹³ C-NMR spectrum (DMSO-d₆) δ ppm: 11.38, 22.54, 26.11, 27.04, 28.34,28.50, 37.06, 38.36, 42.91, 43.45, 49.36, 61.06, 70.11, 73.63, 76.69,76.77, 86.91, 103.00, 112.42, 115.15, 126.35, 130.36, and 142.76.

IR spectrum (KBr) cm⁻¹ : 3376, 2920, 1514, 1450, 1354, 1276, 1076, and1018.

MS spectrum (FAB, positive): 450 (M⁺).

Silica gel TLC: Rf=0.54 (CHCl₃ :MeOH=3:1).

Solubility: 1.0 mg/2-3 ml (H₂ O).

EXAMPLE 5

a)2,3,6,2',3',4',6'-Hepta-O-acetyl-1-O-(2,3-dibenzyloxy-1,3,5(10)-estratriene-17β-yl)-β-maltoside

Ag₂ CO₃ (7.07 g, 25.6 mmol) was added to a solution of2-hydroxyestradiol 2,3-dibenzyl ether (3.00 g, 6.40 mmol) obtained bythe process described in Example 4-a in benzene (300 ml), and thesuspension was distilled until 160 ml of benzene had been removed over 1hour to remove moisture. A solution of acetobromomaltose (13.4 g, 19.2mmol) in benzene (300 ml) was added dropwise to the stirred mixture over1.5 hours and an equal volume of benzene was simultaneously distilled togive a constant volume of the reaction mixture. Distillation wascontinued for further 1 hour, during which benzene was added dropwise tomaintain the volume of the suspension. An insoluble material was removedoff by filtration through a pad of Celite and washed with benzene. Thefiltrate and washings were combined and evaporated in vacuo to obtain acrude product, which was, in turn, subjected to silica gelchromatography (elution solvent, hexane:AcOEt=1.5:1) to afford 4.22 g(60.7%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.72 (3H, s, 18-CH₃), 0.9-2.9 (36H, m,estrogen CH and CH₂, and OCOCH₃ x 7), 4.58 (1H, d, J=8 Hz, pyranose C₁--H), 3.5-4.5 and 4.7-5.6 (14H, m, C₁₇ --H, and pyranose CH--O-- and CH₂--O--), 5.11 (4H, s, C₆ H₅ CH₂ x 2), 6.67 (1H, s, Ar C₄ --H), 6.89 (1H,s, Ar C₁ --H), and 7.2-7.6 (10H, m, C₆ H₅ x 2).

IR spectrum (KBr) cm⁻¹ : 2928, 1754, 1504, 1370, 1230, 1032, 736, and698.

MS spectrum (EI/DI) m/z: 1086 (M⁺).

Silica gel TLC: Rf=0.45 (hexane:AcOEt=1:1).

b) 1-O-(2,3-Dibenzyloxy-1,3,5(10)-estratriene-17β-yl)-β-maltoside

To2,3,6,2',3',4',6'-hepta-O-acetyl-1-O-(2,3-dibenzyloxy-1,3,5(10)-estratriene-17β-yl)-β-maltoside(3.87 g, 3.56 mmol) obtained by the process described in Example 5-a,MeOH (300 ml) was added. After the mixture was mildly heated to obtain ahomogeneous solution, 1N-NaOH (100 ml) was added to it, and the mixturewas stirred at room temperature for 1 hour. After removal of MeOH byevaporation, the residue was extracted with CHCl₃. The organic layer waswashed with brine, and dried over anhydrous Na₂ SO₄, and thenconcentrated in vacuo to obtain a crude product (3.81 g), which was, inturn, subjected to silica gel chromatography (elution solvent, CHCl₃:MeOH=5:1) to afford 1.75 g (62.1%) of the titled compound.

¹ H-NMR spectrum (CD₃ OD) δ ppm: 0.81 (3H, s, 18-CH₃), 0.9-2.8 (15H, m,estrogen CH and CH₂), 3.1-4.1 (13H, m, C₁₇ --H, and pyranose CH--O-- andCH₂ --O--), 4.33 (1H, d, J=8 Hz, pyranose C₁ --H), 4.93 (4H, br.s, C₆ H₅CH₂ x 2), 5.19 (1H, d, J=3 Hz, pyranose C_(1') --H), 6.58 (1H, s, Ar C₄--H), 6.80 (1H, s, Ar C₁ --H), and 7.0-7.5 (10H, m, C₆ H₅ x 2).

IR spectrum (KBr) cm⁻¹ : 3396, 2920, 1502, 1452, 1260, 1020, 736, and696.

MS spectrum (FAB, positive): 792 (M⁺).

Silica gel TLC: Rf=0.26 (CHCl₃ :MeOH=5:1).

c) 2-Hydroxyestradiol 17-(β-maltoside)

To a solution of1-O-(2,3-dibenzyloxy-1,3,5(10)-estratriene-17β-yl)-β-maltoside (1.66 g,2.09 mmol) obtained by the process described in Example 5-b in MeOH (160ml), 5% Pd/C (850 mg) was added, and the mixture was stirred underbubbling of H₂ for 7 hours. The catalyst Pd/C was removed off byfiltration through a pad of Celite. The filtrate was evaporated in vacuoto obtain a crude product (1.18 g). After the crude product wasdissolved in hot MeOH (20 ml), hot CHCl₃ (40 ml) was added to thissolution and an insoluble material was removed off by filtration. To theresulting filtrate, CHCl₃ (100 ml) was added, and the precipitated whitesolid was collected by filtration and dried in vacuo to afford 711 mg(55.5%) of the titled compound.

¹ H-NMR spectrum (CD₃ OD) δ ppm: 0.86 (3H, s, 18-CH₃), 1.0-2.8 (15H, m,estrogen CH and CH₂), 3.1-4.1 (13H, m, C₁₇ --H, and pyranose CH--O-- andCH₂ --O--), 4.38 (1H, d, J=8 Hz, pyranose C₁ --H), 5.17 (1H, d, J=3 Hz,pyranose C_(1') --H), 6.45 (1H, s, Ar C₄ --H), and 6.69 (1H, s, Ar C₁--H).

¹³ C-NMR spectrum (DMSO-d₆) δ ppm: 11.32, 22.43, 26.17, 27.04, 28.28,28.39, 37.01, 38.52, 42.91, 43.40, 49.30, 60.74, 69.89, 72.39, 73.25,75.09, 76.50, 79.05, 79.59, 87.01, 100.61, 102.84, 112.42, 115.51,126.24, 130.30, and 142.82.

IR spectrum (KBr) cm⁻¹ : 3376, 2920, 1514, 1450, 1354, 1274, and 1028.

MS spectrum (FAB, positive): 635 (M⁺ +Na).

Reverse phase TLC: Rf=0.41 (MeOH: H₂ O=3:2).

Solubility: 1.0 mg/2 ml (H₂ O).

EXAMPLE 6

a) 2-Acetylestradiol 17-acetate 3-methoxymethyl ether

NaH (about 60% oil suspension, 1.01 g) was washed with hexane by 3times, and tetrahydrofuran (THF, 40 ml) was added. Chloromethyl methylether (3.83 ml, 50.4 mmol), and 2-acetylestradiol 17-acetate (6.00 g,16.8 mmol) obtained by the process described in Example 1-b in THF (70ml) were added to the NaH suspension. After the mixture was stirred at50° C. for 20 minutes, it was poured onto ice (100 g). Water (600 ml)was added to the mixture. The precipitate was collected by filtration,washed with water, and then dried in vacuo to afford 6.76 g (100%) ofthe titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.82 (3H, s, 18-CH₃), 1.2-3.0 (15H, m,estrogen CH and CH₂), 2.05 (3H, s, C₁₇ --OCOCH₃), 2.62 (3H, s, C₂--COCH₃), 3.50 (3H, s, O--CH₃), 4.68 (1H, t like, C₁₇ --H), 5.24 (2H, s,O--CH₂ --O), 6.88 (1H, s, Ar C₄ --H), and 7.66 (1H, s, Ar C₁ --H).

Silica gel TLC: Rf=0.32 (hexane:AcOEt=4:1).

b) 2-Hydroxyestradiol 2,17-diacetate 3-methoxymethyl ether

A mixture of 2-acetylestradiol 17-acetate 3-methoxymethyl ether (2.50 g,6.24 mmol) obtained by the process described in Example 6-a,m-chloroperbenzoic acid (2.15 g, 12.5 mmol), Na₂ HPO₄ (1.77 g, 12.5mmol), and CHCl₃ (50 ml) was stirred at room temperature for 16 hours.After removed an insoluble material by filtration, the filtrate waspoured into ice-cooled ether. The organic layer was washed successivelywith 5% Na₂ CO₃ solution, water, and brine, thereafter dried overanhydrous Na₂ SO₄, and evaporated in vacuo to obtain a crude product(2.90 g). A part (1.80 g) of the crude product was, in turn, subjectedto silica gel chromatography (elution solvent, hexane:AcOEt=4:1) toafford 1.41 g (87.6%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.81 (3H, s, 18-CH₃), 1.2-3.0 (15H, m,estrogen CH and CH₂), 2.05 (3H, s, C₁₇ --OCOCH₃), 2.30 (3H, s, C₂--OCOCH₃), 3.46 (3H, s, O--CH₃), 4.69 (1H, t like, C₁₇ --H), 5.12 (2H,s, O--CH₂ --O), 6.90 (1H, s, Ar C₄ --H), and 6.93 (1H, s, Ar C₁ --H).

Silica gel TLC: Rf=0.36 (hexane:AcOEt=4:1).

EXAMPLE 7

a) 2-Acetylestradiol 17-acetate 3-benzyl ether

To a solution of 2-acetylestradiol 17-acetate (15.0 g, 42.1 mmol)obtained by the process described in Example 1-b in EtOH (200 ml), K₂CO₃ (8.73 g) and benzyl chloride (7.27 ml) were added, and the mixturewas refluxed for 3 hours. Additional amounts of K₂ CO₃ (5.82 g) andbenzyl chloride (4.84 ml) were added, and the mixture was refluxed forfurther 1.5 hours. After the reaction mixture was cooled to roomtemperature, it was then filtered to remove an insoluble material. Thefiltrate was concentrated in vacuo and a residue was dissolved in CHCl₃(500 ml). The CHCl₃ solution was washed with brine, dried over anhydrousNa₂ SO₄, and then evaporated in vacuo to afford an oily residue.

A mixture of the oily residue obtained above, pyridine (150 ml), andacetic anhydride (55 ml) was stirred overnight at room temperature. Thereaction mixture was poured onto ice (1500 g), and the precipitate wascollected by filtration, washed with water, and then dried in vacuo toafford 18.7 g (99.6%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.82 (3H, s, 18-CH₃), 1.1-3.0 (15H, m,CH and CH₂), 2.05 (3H, s, C₁₇ --OCOCH₃), 2.57 (3H, s, C₂ --COCH₃), 4.69(1H, t like, C₁₇ --H), 5.11 (2H, s, C₆ H₅ CH₂), 6.74 (1H, s, C₄ --H),7.39 (5H, m, C₆ H₅), and 7.71 (1H, s, C₁ --H).

Silica gel TLC: Rf=0.33 (hexane:AcOEt=5:1).

b) 2-Hydroxyestradiol 2,17-diacetate 3-benzyl ether

A mixture of 2-acetylestradiol 17-acetate 3-benzyl ether (1.00 g, 2.24mmol) obtained by the process described in Example 7-a,m-chloroperbenzoic acid (0.773 g, 4.48 mmol), Na₂ HPO₄ (0.318 g, 2.24mmol), and CHCl₃ (25 ml) was stirred at room temperature for 20 hours.The reaction mixture was poured into ether (150 ml). The organic layerwas washed successively with 5% NaOH solution, water, and brine,thereafter it was dried over anhydrous Na₂ SO₄ and evaporated in vacuoto obtain a crude product (1.02 g), which was, in turn, subjected tosilica gel chromatography (elution solvent, CHCl₃ ) to afford 838 mg(81%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.82 (3H, s, 18-CH₃), 1.1-3.0 (15H, m,CH and CH₂), 2.05 (3H, s, C₁₇ --OCOCH₃), 2.25 (3H, s, C₂ --OCOCH₃), 4.68(1H, t like, C₁₇ --H), 5.04 (2H, s, C₆ H₅ CH₂), 6.71 (1H, s, C₄ --H),6.95 (1H, s, C₁ --H), and 7.36 (5H, br.s, C₆ H₅).

Silica gel TLC: Rf=0.49 (CHCl₃).

EXAMPLE 8

a) 2-Acetylestradiol 17-acetate 3-t-butyldimethylsilyl ether

A mixture of 2-acetylestradiol 17-acetate (5.00 g, 14.0 mmol) obtainedby the process described in Example 1-b, t-butyldimethylsilyl chloride(3.18 g, 21.1 mmol), imidazole (2.87 g, 42.2 mmol), anddimethylformamide (30 ml) was stirred at room temperature for 20 hours.The reaction mixture was poured into water and extracted with CHCl₃. Theorganic layer was washed with brine, dried over anhydrous Na₂ SO₄, andthen evaporated in vacuo to obtain a crude product, which was, in turn,subjected to silica gel chromatography (elution solvent, CHCl₃) toafford 6.64 g (101%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.25 [6H, s, Si(CH₃)₂ ], 0.83 (3H, s,18-CH₃), 1.00 (9H, s, Si-t-C₄ H₉), 1.1-3.0 (15H, m, estrogen CH andCH₂), 2.05 (3H, s, C₁₇ --OCOCH₃), 2.58 (3H, s, C₂ --COCH₃), 4.68 (1H, tlike, C₁₇ --H), 6.57 (1H, s, Ar C₄ --H), and 7.55 (1H, s, Ar C₁ --H).

Silica gel TLC: Rf=0.39 (CHCl₃).

b) 2-Hydroxyestradiol 2,17-diacetate 3-t-butyldimethylsilyl ether

A mixture of 2-acetylestradiol 17-acetate 3-t-butyldimethylsilyl ether(1.00 g, 2.12 mmol) obtained by the process described in Example 8-a,m-chloroperbenzoic acid (0.733 g, 4.25 mmol), Na₂ HPO₄ (0.602 g, 4.24mmol), and CHCl₃ (25 ml) was stirred at room temperature for 19 hours.The reaction mixture was poured into ether (300 ml). Tile organic layerwas washed successively with 5% Na₂ CO₃ solution, water, and brine,thereafter it was dried over anhydrous Na₂ SO₄ and evaporated in vacuoto obtain a crude product, which was, in turn, subjected to silica gelchromatography (elution solvent, CHCl₃) to afford 898 mg (87%) of thetitled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.19 [6H, s, Si(CH₃)₂ ], 0.82 (3H, s,18-CH₃), 0.98 (9H, s, Si-t-C₄ H₉), 1.2-2.9 (15H, m, estrogen CH andCH₂), 2.05 (3H, s, C₁₇ --OCOCH₃), 2.26 (3H, s, C₂ --OCOCH₃), 4.68 (1H, tlike, C₁₇ --H), 6.59 (1H, s, Ar C₄ --H), and 6.90 (1H, s, Ar C₁ --H).

Silica gel TLC: Rf=0.50 (CHCl₃).

EXAMPLE 9

a) 2-Acetylestradiol diacetate

A mixture of 2-acetylestradiol 17-acetate (1.00 g, 2.81 mmol) obtainedby the process described in Example 1-b, pyridine (6ml), and aceticanhydride (3 ml) was stirred overnight at room temperature. The reactionmixture was poured onto ice (50 g), and the precipitate was collected byfiltration, washed with water, and then dried in vacuo to afford 1.11 g(99.1%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.84 (3H, s, 18-CH₃), 1.1-3.0 (15H, m,estrogen CH and CH₂), 2.07 (3H, s, C₁₇ --OCOCH₃), 2.34 (3H, s, C₃--OCOCH₃), 2.53 (3H, s, C₂ --COCH₃), 4.70 (1H, t like, C₁₇ --H), 6.81(1H, s, Ar C₄ --H), and 7.74 (1H, s, Ar C₁ --H).

Silica gel TLC: Rf=0.21 (CHCl₃).

b) 2-Hydroxyestradiol triacetate

A mixture of 2-acetylestradiol diacetate (100 mg, 0.251 mmol) obtainedby the process described in Example 9-a, m-chloroperbenzoic acid (0.130g, 0.753 mmol), Na₂ HPO₄ (71.3 mg, 0.502 mmol), and CHCl₃ (2 ml) wasstirred at room temperature for 40 hours. The reaction mixture waspoured into ether (50 ml). The organic layer was washed successivelywith 5% Na₂ CO₃ solution, water, and brine, thereafter it was dried overanhydrous Na₂ SO₄ and evaporated in vacuo to obtain a crude product,which was, in turn, subjected to silica gel thin-layer chromatography(developing solvent, CHCl₃ :MeOH=100:1) to afford 92.9 mg (89%) of thetitled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.82 (3H, s, 18-CH₃), 1.1-3.0 (15H, m,estrogen CH and CH₂), 2.05 (3H, s, C₁₇ --OCOCH₃), 2.27 (6H, s, C₂--OCOCH₃ and C₃ --OCOCH₃), 4.68 (1H, t like, C₁₇ --H), 6.87 (1H, s, ArC₄ --H), and 7.05 (1H, s, Ar C₁ --H).

Silica gel TLC: Rf=0.37 (CHCl₃).

EXAMPLE 10

a) 2-Hydroxyestradiol 17-acetate 3-methoxymethyl ether

To a solution of 2-hydroxyestradiol 2,17-diacetate 3-methoxymethyl ether(1.35 g, 3.24 mmol) obtained by the process described in Example 6-b inacetone (32.3 ml), a solution of KHCO₃ (647 mg, 6.46 mmol) in water(16.1 ml) and MeOH (80.7 ml) was added, and the mixture was stirred atroom temperature for 22 hours and then 50° C. for 3 hours. After water(20 ml) was added to the reaction mixture, acetone and MeOH were removedby evaporation. The precipitate was collected by filtration, washed withwater, and then dried in vacuo to afford 1.20 g (99.2%) of the titledcompound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.82 (3H, s, 18-CH₃), 1.2-2.9 (15H, m,estrogen CH and CH₂), 2.05 (3H, S, C₁₇ --OCOCH₃), 3.51 (3H, s, O--CH₃),4.68 (1H, t like, C₁₇ --H), 5.16 (2H, s, O--CH₂ --O), 5.74 (1H, s, C₂--OH), 6.79 (1H, s, Ar C₄ --H), and 6.89 (1H, s, Ar C₁ --H).

Silica gel TLC: Rf=0.49 (hexane:AcOEt=2:1).

b) 2-Benzyloxyestradiol 17-acetate 3-methoxymethyl ether

To a solution of 2-hydroxyestradiol 17-acetate 3-methoxymethyl ether(1.13 g, 3.02 mmol) obtained by the process described in Example 10-a inEtOH (25 ml), k₂ CO₃ (626 mg, 4.54 mmol) and benzyl chloride (0.452 ml,3.93 mmol) were added and the mixture was refluxed for 4 hours.Additional amount of benzyl chloride (0.174 ml, 1.51 mmol) was added,and the mixture was refluxed for further 2 hours. After the reactionmixture was cooled to room temperature, it was then filtered to removean insoluble material. The filtrate was evaporated in vacuo and aresidue was mixed with water to obtain a precipitate. The precipitatewas collected by filtration, washed with water, and then dried in vacuoto afford a mixture (1.44 g) of 2-benzyloxyestradiol 3-methoxymethylether and 2-benzyloxyestradiol 17-acetate 3-methoxymethyl ether.

A part (1.41 g) of the mixture of 2-benzyloxyestradiol 3-methoxymethylether and 2-benzyloxyestradiol 17-acetate 3-methoxymethyl ether obtainedabove was treated with pyridine (4.0 ml, 49.5 mmol) and acetic anhydride(3.0 ml, 31.7 mmol) for 23 hours at room temperature. The reactionmixture was poured onto ice (150 g), and 2N-HCl (40 ml) was added to it.After extraction with AcOEt, the organic layer was washed successivelywith 1N-HCl, saturated NaHCO₃ solution, water, and brine, thereafter itwas dried over anhydrous Na₂ SO₄ and evaporated in vacuo to obtain acrude product (1.51 g), which was, in turn, subjected to silica gelchromatography (elution solvent, hexane:AcOEt=6:1) to afford 1.35 g(98.5%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.81 (3H, s, 18-CH₃), 1.1-2.9 (15H, m,estrogen CH and CH₂), 2.05 (3H, s, C₁₇ --OCOCH₃), 3.51 (3H, s, O--CH₃),4.68 (1H, t like, C₁₇ --H), 5.10 (2H, s, C₆ H₅ CH₂), 5.18 (2H, s, O--CH₂--O), 6.85 (2H, s, Ar C₄ --H and C₁ --H), and 7.2-7.5 (5H, m, C₆ H₅).

Silica gel TLC: Rf=0.50 (hexane:AcOEt=4:1).

c) 2-Benzyloxyestradiol 17-acetate

A mixture of 2-benzyloxyestradiol 17-acetate 3-methoxymethyl ether (3.70g, 7.97 mmol) obtained by the process described in Example 10-b, aceticacid (20 ml), and 2N-HCl (2.0 ml) was stirred at room temperature for1.5 hours. The reaction mixture was poured into water (500 ml), and theprecipitate was collected by filtration, washed with water, and thendried in vacuo to obtain a crude product (3.41 g), which was, in turn,subjected to silica gel chromatography (elution solvent,hexane:AcOEt=6:1) to afford 3.02 g (90.1%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.83 (3H, s, 18-CH₃), 1.1-2.9 (15H, m,estrogen CH and CH₂), 2.05 (3H, s, C₁₇ --OCOCH₃), 4.69 (1H, t like, C₁₇--H), 5.06 (2H, s, C₆ H₅ CH₂), 6.65 (1H, s, Ar C₄ --H), 6.85 (1H, s, ArC₁ --H), and 7.39 (5H, s, C₆ H₅).

Silica gel TLC: Rf=0.37 (hexane:AcOEt=5:1).

EXAMPLE 11

a) 2-Hydroxyestradiol 3-methoxymethyl ether

A mixture of 2-hydroxyestradiol 2,17-diacetate 3-methoxymethyl ether(5.30 g, 12.7 mmol) obtained by the process described in Example 6-b,MeOH (106 ml), and 1N--NaOH (53 ml) was stirred at room temperature for2.5 hours. After MeOH was removed by evaporation, acetic acid (2.37 ml)was added to the residue. After extraction with AcOEt, the organic layerwas washed successively with saturated NaHCO₃ solution, water, andbrine, thereafter it was dried over anhydrous Na₂ SO₄ and evaporated invacuo to afford 4.23 g (100%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.77 (3H, s, 18-CH₃), 1.1-2.9 (15H, m,estrogen CH and CH₂), 3.51 (3H, s, O--CH₃), 3.73 (1H, t like, C₁₇ --H),5.16 (2H, s, O--CH₂ --O), 5.75 (1H, br.s, C₂ --OH), 6.79 (1H, s, Ar C₄--H), and 6.89 (1H, s, Ar C₁ --H).

Silica gel TLC: Rf=0.20 (hexane:AcOEt=2:1).

b) 2-Benzyloxyestradiol 3-methoxymethyl ether

To a solution of 2-hydroxyestradiol 3-methoxymethyl ether (4.20 g, 12.6mmol) obtained by the process described in Example 11-a in EtOH (100ml), K₂ CO₃ (2.62 g, 19.0 mmol) and benzyl chloride (1.89 ml, 16.4 mmol)were added, and the mixture was refluxed for 4 hours, Additional amountof benzyl chloride (0.73 ml, 6.33 mmol) was added, and the mixture wasrefluxed for further 2 hours. After the reaction mixture was cooled toroom temperature, it was then filtered to remove an insoluble material.The filtrate was concentrated in vacuo and a residue was mixed withwater to obtain a precipitate. The precipitate was collected byfiltration, washed with water, and then dried in vacuo to obtain a crudeproduct (5.64 g).

A part (1.50 g) of the crude product was subjected to silica gelchromatography (elution solvent, hexane:AcOEt=3:1) to afford 1.40 g(98.6%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.76 (3H, s, 18-CH₃), 1.0-2.9 (15H, m,estrogen CH and CH₂), 3.51 (3H, s, O--CH₃), 3.72 (1H, t like, C₁₇ --H),5.10 (2H, s, C₆ H₅ CH₂), 5.19 (2H, s, O--CH₂ --O), 6.85 (1H, s, Ar C₄--H), 6.88 (1H, s, Ar C₁ --H), and 7.2-7.5 (5H, m, C₆ H₅).

Silica gel TLC: Rf=0.38 (hexane:AcOEt=2:1).

c) 2-Benzyloxyestradiol 17-acetate 3-methoxymethyl ether

A mixture of another part (4.00 g) of the crude product of2-benzyloxyestradiol 3-methoxymethyl ether obtained by the processdescribed in Example 11-b, pyridine (12 ml, 148 mmol), and aceticanhydride (9.0 ml, 95.2 mmol) was stirred for 23 hours at roomtemperature. The reaction mixture was poured onto ice (200 g) and 2N-HCl(200 ml) was added to it. After extraction with AcOEt, the organic layerwas washed successively with 1N-HCl, saturated NaHCO₃ solution, water,and brine, thereafter it was dried over anhydrous Na₂ SO₄ and evaporatedin vacuo to obtain a crude product (4.36 g), which was, in turn,subjected to silica gel chromatography (elution solvent,hexane:AcOEt=6:1) to afford 3.93 g (94.7% from 2-hydroxyestradiol3-methoxymethyl ether) of the titled compound.

This compound showed the same physico-chemical data to those describedin Example 10-b.

This compound can be converted into 2-benzyloxyestradiol 17-acetate bythe process described in Example 10-c.

EXAMPLE 12

a) 2-Benzyloxyestradiol

A mixture of the purified 2-benzyloxyestradiol 3-methoxymethyl ether(1.35 g, 3.19 mmol) obtained by the process described in Example 11-b,MeOH (27 ml), and concentrated HCl (2.7 ml) was stirred for 2 hours atroom temperature. After removal of a half volume of MeOH by evaporation,the residue was poured into water (300 ml). The precipitate wascollected by filtration, washed with water, and then dried in vacuo toafford 1.20 g (99.2%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.78 (3H, s, 18-CH₃), 1.0-2.9 (15H, m,estrogen CH and CH₂), 3.73 (1H, t like, C₁₇ --H), 5.07 (2H, s, C₆ H₅CH₂), 6.66 (1H, s, Ar C₄ --H), 6.87 (1H, s, Ar C₁ --H), and 7.40 (5H, s,C₆ H₅).

Silica gel TLC: Rf=0.37 (hexane:AcOEt=2:1).

b) 2-Benzyloxyestradiol diacetate

A mixture of 2-benzyloxyestradiol (1.18 g, 3.12 mmol) obtained by theprocess described in Example 12-a, pyridine (3.0 ml, 37.1 mmol), andacetic anhydride (2.5 ml, 26.4 mmol) was stirred for 21 hours at roomtemperature. The reaction mixture was poured onto ice and theprecipitate was collected by filtration, washed with water, and thendried in vacuo to afford 1.40 g (97.2%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.82 (3H, s, 18-CH₃), 1.1-2.9 (15H, m,estrogen CH and CH₂), 2.06 (3H, s, C₁₇ --OCOCH₃), 2.26 (3H, s, C₃--OCOCH₃), 4.68 (1H, t like, C₁₇ --H), 5.04 (2H, s, C₆ H₅ CH₂), 6.75(1H, s, Ar C₄ --H), 6.93 (1H, s, Ar C₁ --H), and 7.36 (5H, s, C₆ H₅).

Silica gel TLC: Rf=0.42 (hexane:AcOEt=4:1).

c) 2-Benzyloxyestradiol 17-acetate

To a solution of 2-benzyloxyestradiol diacetate (1.35 g, 2.92 mmol)obtained by the process described in Example 12-b in acetone (29.2 ml),a solution of KHCO₃ (585 mg) in water (14.6 ml) and MeOH (73.0 ml) wasadded, and the mixture was stirred at room temperature for 21 hours,followed at 50° C. for 6 hours. After water was added to the reactionmixture, acetone and MeOH were removed by evaporation. The precipitatewas collected by filtration, washed with water, and then dried in vacuoto obtain a crude product (1.23 g), which was, in turn, subjected tosilica gel chromatography (elution solvent, hexane:AcOEt=6:1) to afford1.18 g (95.9%) of the titled compound.

This compound showed the same physico-chemical data to those describedin Example 10-c.

EXAMPLE 13

a)2,3,4,6-Tetra-O-acetyl-1-O-(17β-acetoxy-2-benzyloxy-1,3,5(10)-estratriene-3-yl)-β-D-glucopyranoside

CdCO₃ (6.55 g, 38.0 mol) was added to a solution of 2-benzyloxyestradiol17-acetate (4.00 g, 9.49 mmol) obtained by the process described inExample 10-c in toluene (250 ml), and the suspension was distilled until50 ml of toluene had been removed. A solution of acetobromoglucose (11.7g, 28.5 mmol) in toluene (300 ml) was added dropwise to the stirredmixture over 6 hours and an equal volume of toluene was simultaneouslydistilled to give a constant volume of the reaction mixture.Distillation was continued for further 15 hours during which generatedmoisture was removed. Additional amounts of CdCO₃ (6.55 g) andacetobromoglucose (11.7 g) were added, and the mixture was refluxed forfurther 18 hours during which generated moisture was removed. Aninsoluble material was removed off by filtration through a pad ofCelite. The filtrate was evaporated in vacuo to obtain a crude product,which was, in turn, subjected to silica gel chromatography (elutionsolvent, hexane:AcOEt=2:1) to afford 4.30 g (60.3%) of the titledcompound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.82 (3H, s, 18-CH₃), 1.1-2.9 (30H, m,estrogen CH and CH₂, and OCOCH₃ x 5), 3.78 (1H, m, pyranose C₅ --H),4.0-4.4 (2H, m, pyranose C₆ --H₂), 4.69 (1H, t like, C₁₇ --H), 4.9-5.4(6H, m, pyranose C₁ --H, C₂ --H, C₃ --H, C₄ --H, and C₆ H₅ CH₂), 6.83(1H, s, Ar C₄ --H or C₁ --H), 6.88 (1H, s, Ar C₁ --H or C₁ --H), and7.2-7.5 (5H, m, C₆ H₅).

Silica gel TLC: Rf=0.26 (hexane:AcOEt=2:1).

b)1-O-(2-Benzyloxy-17β-hydroxy-1,3,5(10)-estratriene-3-yl)-β-D-glucopyranoside

To2,3,4,6-tetra-O-acetyl-1-O-(17β-acetoxy-2-benzyloxy-1,3,5(10)-estratriene-3-yl)-β-D-glucopyranoside(4.20 g, 5.59 mmol) obtained by the process described in Example 13-a,MeOH (80 ml) and 1N-NaOH (40 ml) were added. The mixture was stirred atroom temperature for 1.5 hours and then at 45° C. for 1 hour. Afterremoval of MeOH by evaporation, an aqueous layer was separated from anoily residue by decantation. The aqueous layer was extracted with AcOEt.The AcOEt layer and the oily residue were combined and concentrated invacuo to obtain a crude product (2.71 g), which was, in turn, subjectedto silica gel chromatography (elution solvent, CHCl₃ :MeOH=8:1) toafford 2.25 g (74.5%) of the titled compound.

¹ H-NMR spectrum (CD₃ OD) δ ppm: 0.73 (3H, s, 18-CH₃), 1.0-2.8 (15H, m,estrogen CH and CH₂), 3.3-3.9 (7H, m, C₁₇ --H, and pyranose C₂ --H, C₃--H, C₄ --H, C₅ --H and C₆ --H₂), 4.84-4.91 (1H, m, pyranose C₁ --H),5.06 (2H, s, C₆ H₅ CH₂), 6.88 (2H, s, Ar C₄ --H and C₁ --H), and 7.2-7.5(5H, m, C₆ H₅).

Silica gel TLC: Rf=0.55 (CHCl₃ :MeOH=5:1).

c) 2-Hydroxyestradiol 3-(β-D-glucopyranoside)

To a solution of1-O-(2-benzyloxy-17β-hydroxy-1,3,5(10)-estratriene-3-yl)-β-D-glucopyranoside(2.03 g, 3.75 mmol) obtained by the process described in Example 13-b inMeOH (100 ml), a suspension of 5% Pd/C (0.50 g) in MeOH (50 ml) wasadded, and the mixture was stirred under bubbling of H₂ for 2 hours. Thecatalyst Pd/C was removed off by filtration through a pad of Celite. Thefiltrate was evaporated in vacuo to obtain a crude product (1.65 g),which was, in turn, subjected to silica gel chromatography (elutionsolvent, CHCl₃ :MeOH=5:1) to afford 1.57 g (92.9%) of the titledcompound.

¹ H-NMR spectrum (CD₃ OD) δ ppm: 0.74 (3H, s, 18-CH₃), 1.0-2.8 (15H, m,estrogen CH and CH₂), 3.3-4.0 (7H, m, C₁₇ --H, and pyranose C₂ --H, C₃--H, C₄ --H, C₅ --H and C₆ --H₂), 4.65-4.73 (1H, m, pyranose C₁ --H),6.77 (1H, s, Ar C₄ --H), and 6.85 (1H, s, Ar C₁ --H).

¹³ C-NMR spectrum (DMSO-d₆) δ ppm: 11.11, 22.71, 25.95, 26.98, 28.50,29.85, 36.57, 38.41, 42.69, 43.62, 49.52, 60.79, 69.84, 73.30, 75.85,77.10, 79.97, 103.00, 112.59, 117.62, 126.73, 134.75, 143.04, and144.66.

IR spectrum (KBr) cm⁻¹ : 3408, 2920, 2868, 1588, 1502, 1436, 1408, 1384,1352, 1282, 1210, 1068, 1040, 886, 864, 830, and 810.

Silica gel TLC: Rf=0.24 (CHCl₃ :MeOH=5:1).

Solubility: 1.0 mg/3 ml (H₂ O).

EXAMPLE 14

a)2,3,4,6-Tetra-O-acetyl-1-O-(2-acetyl-17β-acetoxy-1,3,5(10)-estratriene-3-yl)-β-D-glucopyranosideand2,3,4,6-tetra-O-acetyl-1-O-(2-acetyl-17β-acetoxy-1,3,5(10)-estratriene-3-yl)-α-D-glucopyranoside

CdCO₃ (1.72 g, 10.0 mmol) was added to a solution of 2-acetylestradiol17-acetate (891 mg, 2.50 mmol) obtained by the process described inExample 1-b in toluene (50 ml), and the suspension was distilled until15 ml of toluene had been removed. A solution of acetobromoglucose (3.08g, 7.50 mmol) in toluene (50 ml) was added dropwise to the stirredmixture over 2 hours and an equal volume of toluene was simultaneouslydistilled to give a constant volume of the reaction mixture.Distillation was continued for further 2 hours during which toluene wasadded dropwise to maintain the volume of the suspension. Sincepreliminary TLC analysis showed that the starting material had not beencompletely consumed, CdCO₃ (0.86 g, 5.00 mmol) was added to thesuspension and subsequently a solution of acetobromoglucose (1.54 g,3.75 mmol) in toluene (30 ml) was further added dropwise over 1 hour,and an equal volume of toluene was simultaneously distilled to give aconstant volume of the reaction mixture. Distillation was continued forfurther 1.5 hours during which toluene was added dropwise to maintainthe volume of the suspension.

An insoluble material was removed off by filtration through a pad ofCelite. The filtrate was evaporated in vacuo to obtain a crude product,which was, in turn, subjected to silica gel chromatography (elutionsolvent, hexane:AcOEt=2:1) to afford 662 mg (38.5%) of2,3,4,6-tetra-O-acetyl-1-O-(2-acetyl-17β-acetoxy-1,3,5(10)-estratriene-3-yl)-β-D-glucopyranosideand 1.23 g (purity 67%, net weight 824 mg, yield 47.9%) of2,3,4,6-tetra-O-acetyl-1-O-(2-acetyl-17β-acetoxy-1,3,5(10)-estratriene-3-yl-α-D-glucopyranoside.

Physico-chemical data;

2,3,4,6-tetra-O-acetyl-1-O-(2-acetyl-17β-acetoxy-1,3,5(10)-estratriene-3-yl)-β-D-glucopyranoside

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.82 (3H, s, 18-CH₃), 1.2-3.0 (30H, m,estrogen CH and CH₂, and OCOCH₃ x 5), 2.52 (3H, s, C₂ --COCH₃), 3.8-4.3(3H, m, pyranose C₅ --H and C₆ --H₂), 4.68 (1H, t like, C₁₇ --H),5.1-5.4 (4H, m, pyranose C₁ --H, C₂ --H, C₃ --H and C₄ --H), 6.76 (1H,s, Ar C₄ --H), and 7.61 (1H, s, Ar C₁ --H).

Silica gel TLC: Rf=0.12 (hexane:AcOEt=2:1).

2,3,4,6-tetra-O-acetyl-1-O-(2-acetyl-17β-acetoxy-1,3,5(10)-estratriene-3-yl)-α-D-glucopyranoside

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.83 (s, 18-CH₃), 1.2-3.0 (m, estrogenCH and CH₂, and OCOCH₃ x 5), 2.73 (s, C₂ --COCH₃), 3.8-4.4 (m, pyranoseC₅ --H and C₆ --H₂), 4.68 (t like, C₁₇ --H), 5.0-5.8 (m, pyranose C₁--H, C₂ --H, C₃ --H and C₄ --H), 6.96 (s, Ar C₄ --H), and 7.68 (s, Ar C₁--H).

Silica gel TLC: Rf=0.18 (hexane:AcOEt=2:1).

b)2,3,4,6-Tetra-O-acetyl-1-O-(2,17β-diacetoxy-1,3,5(10)-estratriene-3-yl)-β-D-glucopyranoside

A mixture of2,3,4,6-tetra-O-acetyl-1-O-(2-acetyl-17β-acetoxy-1,3,5(10)-estratriene-3-yl)-β-D-glucopyranoside(395 mg, 0.575 mmol) obtained by the process described in Example 14-a,m-chloroperbenzoic acid (298 mg, 1.73 mmol), Na₂ HPO₄ (245 mg, 1.73mmol), and CHCl₃ (8 ml) was stirred at room temperature for 17 hours.After an insoluble material was removed off by filtration, the filtratewas poured into ice-cooled ether. The organic layer was washedsuccessively with 5% Na₂ CO₃ solution, water, and brine, thereafter itwas dried over anhydrous Na₂ SO₄ and evaporated in vacuo to afford 440mg of the titled compound as a crude product.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.80 (3H, s, 18-CH₃), 1.1-3.0 (30H, m,estrogen CH and CH₂, and OCOCH₃ x 5), 2.25 (3H, s, C₂ --OCOCH₃), 3.8-4.4(3H, m, pyranose C₅ --H and C₆ --H₂), 4.68 (1H, t like, C₁₇ --H),5.0-5.4 (4H, m, pyranose C₁ --H, C₂ --H, C₃ --H and C₄ --H), 6.71 (1H,s, Ar C₄ --H), and 6.93 (1H, s, Ar C₁ --H).

Silica gel TLC: Rf=0.56 (CHCl₃ :MeOH=20:1).

c) 2-Hydroxyestradiol 3-(β-D-glucopyranoside)

To the crude product of2,3,4,6-tetra-O-acetyl-1-O-(2,17β-diacetoxy-1,3,5(10)-estratriene-3-yl)-β-D-glucopyranoside(400 mg) obtained by the process described in Example 14-b, MeOH (10 ml)and 1N-NaOH (5 ml) were added. The mixture was stirred at roomtemperature for 1.5 hours. After removal of MeOH by evaporation, water(15 ml) and acetic acid (0.150 ml) were added to precipitate the desiredproduct. The precipitate was collected by filtration and subjected tosilica gel chromatography (elution solvent, CHCl₃ :MeOH=5:1) to afford196 mg [83.1% from2,3,4,6-tetra-O-acetyl-1-O-(2-acetyl-17β-acetoxy-1,3,5(10)-estratriene-3-yl)-β-D-glucopyranoside]of the titled compound.

Physico-chemical data of this compound were the same to those describedin Example 13-c.

EXAMPLE 15

a)2,3,4,6-Tetra-O-acetyl-1-O-(2,17β-diacetoxy-1,3,5(10)-estratriene-3-yl)-α-D-glucopyranoside

A mixture of2,3,4,6-tetra-O-acetyl-1-O-(2-acetyl-17β-acetoxy-1,3,5(10)-estratriene-3-yl)-α-D-glucopyranoside(790 ml) purity 67%, net weight 529 mg, O.771 mmol) obtained by theprocess described in Example 14-a, m-chloroperbenzoic acid (596 mg, 3.45mmol), Na₂ HPO₄ (490 mg, 3.45 mmol), and CHCl₃ (16 ml) was stirred atroom temperature for 22 hours. After an insoluble material was removedoff by filtration, the filtrate was poured into ice-cooled ether. Theorganic layer was washed successively with 5% Na₂ CO₃ solution, water,and brine, thereafter it was dried over anhydrous Na₂ SO₄ and evaporatedin vacuo to afford 863 mg of the titled compound as a crude product.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.82 (s, 18-CH₃), 1.1-2.9 (m, estrogenCH and CH₂, and OCOCH₃ x 5), 2.37 (s, C₂ --OCOCH₃), 3.7-4.4 (m, pyranoseC₅ --H and C₆ --H₂), 4.69 (t like, C₁₇ --H), 4.9-5.8 (m, pyranose C₁--H, C₂ --H, C₃ --H and C₄ --H), 6.91 (s, Ar C₄ --H), and 6.98 (s, Ar C₁--H).

Silica gel TLC: Rf=0.44 (CHCl₃ :MeOH=50:1).

b) 2-Hydroxyestradiol 3-(α-D-glucopyranoside)

To the crude product of2,3,4,6-tetra-O-acetyl-1-O-(2,17β-diacetoxy-1,3,5(10)-estratriene-3-yl)-α-D-glucopyranoside(850 mg) obtained by the process described in Example 15-a, MeOH (20 ml)and 1N-NaOH (10 ml) were added. The mixture was stirred at roomtemperature for 1 hour. After removal of MeOH by evaporation, water (50ml) and acetic acid (0.366 ml) were added to precipitate the desiredproduct. The muddy precipitate was collected by filtration andcrystallized from MeOH to afford 240 mg [70.4% from2,3,4,6-tetra-O-acetyl-1-O-(2-acetyl-17β-acetoxy-1,3,5(10)-estratriene-3-yl)-α-D-glucopyranoside]of the titled compound.

¹ H-NMR spectrum (DMSO-d₆ :CD₃ OD=3:1) δ ppm: 0.71 (3H, s, 18-CH₃ a),1.0-2.8 (15H, m, estrogen CH and CH₂), 3.3-3.9 (7H, m, C₁₇ --H, andpyranose C₂ --H, C₃ --H, C₄ --H, C₅ --H and C₆ --H₂), 5.14 (1H, d, J=3Hz, pyranose C₁ --H), 6.73 (1H, s, Ar C₄ --H), and 6.88 (1H, s, Ar C₁--H).

¹³ C-NMR spectrum (DMSO-d₆) β ppm: 11.16, 22.70, 26.01, 27.04, 28.39,29.91, 36.68, 38.36, 42.75, 43.67, 49.58, 60.68, 69.95, 71.95, 73.14,73.58, 80.02, 100.50, 112.53, 118.01, 126.68, 134.80, 143.04, and145.14.

Silica gel TLC: Rf=0.23 (CHCl₃ :MeOH=5:1).

EXAMPLE 16

a) 2-Hydroxyestradiol 17-acetate 3-benzyl ether

To a solution of 2-hydroxyestradiol 2,17-diacetate 3-benzyl ether (9.00g, 19.4 mmol) obtained by the process described in Example 7-b inacetone (450 ml), a solution of KHCO₃ (9.00 g) in water (225 ml) andMeOH (1125 ml) was added, and the mixture was stirred at roomtemperature for 47 hours. The mixture was poured into ether (6000 ml),and the organic layer was washed successively with water, and brine,thereafter it was dried over anhydrous Na₂ SO₄ and evaporated in vacuoto obtain a crude product (8.15 g), which was, in turn, subjected tosilica gel chromatography (elution solvent, CHCl₃) to afford 7.59 g(92.8%) of the titled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.82 (3H, s, 18-CH₃), 1.1-2.9 (15H, m,CH and CH₂), 2.05 (3H, s, C₁₇ --OCOCH₃), 4.68 (1H, t like, C₁₇ --H),5.05 (2H, s, C₆ H₅ CH₂), 5.50 (1H, s, OH), 6.64 (1H, s, C₄ --H), 6.89(1H, s, C₁ --H), and 7.39 (5H, br.s, C₆ H₅).

Silica gel TLC: Rf=0.36 (CHCl₃).

b)2,3,4,6-Tetra-O-acetyl-1-O-(17β-acetoxy-3-benzyloxy-1,3,5(10)-estratriene-2-yl)-β-D-glucopyranosideCdCO₃ (6.55 g, 38.0 mmol) was added to a solution of 2-hydroxyestradiol17-acetate 3-benzyl ether (4.00 g, 9.51 mmol) obtained by the processdescribed in Example 16-a in benzene (300 ml), and the suspension wasdistilled until 100 ml of benzene had been removed. A solution ofacetobromoglucose (11.7 g, 28.5 mmol) in benzene (300 ml) was addeddropwise to the stirred mixture over 3 hours and an equal volume ofbenzene was simultaneously distilled to give a constant volume of thereaction mixture. Heating was continued further. After 30 minutes,additional amount of CdCO₃ (6.60 g, 38.3 mmol) was added, and after 19hours, additional amounts of CdCO₃ (5.0 g, 29.0 mmol) andacetobromoglucose (5.3 g, 12.9 mmol) in benzene (100 ml) were furtheradded. The mixture was refluxed for further 23 hours, meanwhilegenerated moisture was removed. An insoluble material was removed off byfiltration through a pad of Celite and washed with benzene. The filtrateand washings were combined and evaporated in vacuo to obtain a crudeproduct, which was, in turn, subjected to silica gel chromatography(elution solvent, hexane:AcOEt=2:1) to afford 4.51 g (63.3%) of thetitled compound.

¹ H-NMR spectrum (CDCl₃) δ ppm: 0.82 (3H, s, 18-CH₃), 1.1-2.9 (30H, m,estrogen CH and CH₂, and OCOCH₃ x 5), 3.73 (1H, m, pyranose C₅ --H),4.0-4.4 (2H, m, pyranose C₆ --H₂), 4.68 (1H, t like, C₁₇ --H), 4.9-5.4(6H, m, pyranose C₁ --H, C₂ --H, C₃ --H, C₄ --H, and C₆ H₅ CH₂), 6.67(1H, s, Ar C₄ --H), 7.07 (1H, s, Ar C₁ --H), and 7.2-7.5 (5H, m, C₆ H₅H).

¹³ C-NMR spectrum (DMSO-d₆) δ ppm: 11.81, 19.99, 20.16, 20.26, 20.75,22.70, 25.57, 26.77, 27.14, 28.61, 36.46, 37.93, 42.42, 43.34, 49.03,61.98, 68.27, 70.11, 70.81, 72.00, 81.81, 98.77, 115.08, 115.62, 127.22,127.49, 128.14, 131.55, 132.42, 137.24, 144.01, 146.51, 168.83, 169.20,169.42, 169.80, and 170.18.

IR spectrum (KBr) cm⁻¹ : 2936, 2872, 1756, 1508, 1434, 1372, 1246, 1222,1042, and 906.

MS spectrum (EI/DI) m/z: 750 (M⁺).

Silica gel TLC: Rf=0.27 (hexane:AcOEt=2:1).

c)1-O-(3-Benzyloxy-17β-hydroxy-1,3,5(10)-estratriene-2-yl)-β-D-glucopyranoside

To2,3,4,6-tetra-O-acetyl-1-O-(17β-acetoxy-3-benzyloxy-1,3,5(10)-estratriene-2-yl)-β-D-glucopyranoside(3.80 g, 5.06 mmol) obtained by tile process described in Example 16-b,MeOH (266 ml) was added. After the mixture was mildly heated to obtain ahomogeneous solution, 1N-NaOH (76 ml) was added, and the mixture wasstirred at room temperature for 1.5 hours. After removal of MeOH byevaporation, the residue was extracted with AcOEt. The organic layer waswashed with brine, dried over anhydrous Na₂ SO₄, and concentrated invacuo to obtain a crude product (2.68 g), which was, in turn, subjectedto silica gel chromatography (elution solvent, CHCl₃ :MeOH=7:1) toafford 2.36 g (86.1%) of the titled compound.

¹ H-NMR spectrum (CD₃ OD) δ ppm: 0.77 (3H, s, 18-CH₃). 1.0-2.8 (15H, m,estrogen CH and CH₂). 3.2-4.0 (7H, m, C₁₇ --H, and pyranose C₂ --H, C₃--H, C₄ --H, C₅ --H and C₆ --H). 4.82-4.90 (1H, m, pyranose C₁ --H),5.09 (2H, s, C₆ H₅ CH₂). 6.71 (1H, s, Ar C₄ --H), 7.13 (1H, s, Ar C₁--H), and 7.2-7.6 (5H, m, C₆ H₅).

¹³ C-NMR spectrum (DMSO-d₆) δ ppm: 11.16, 22.70, 25.68, 26.87, 28.50,29.85, 36.68, 38.36, 42.75, 43.67, 49.52, 60.74, 69.79, 70.60, 73.36,76.83, 76.94, 79.97, 101.37, 114.48, 115.78, 127.38, 128.08, 129.87,133.07, 137.62, 145.26, and 146.01.

IR spectrum (KBr) cm⁻¹ : 3400, 2920, 2868, 1504, 1454, 1412, 1382, 1324,1286, 1260, 1210, 1072, 888, 748, and 698.

Silica gel TLC: Rf=0.44 (CHCl₃ :MeOH=5:1).

d) 2-Hydroxyestradiol 2-(β-D-glucopyranoside)

To a solution of1-O-(3-benzyloxy-17β-hydroxy-1,3,5(10)-estratriene-2-yl)-β-D-glucopyranoside(2.30 g, 4.25 mmol) obtained by the process described in Example 16-c inMeOH (250 ml), a suspension of 5% Pd/C (1.00 g) in MeOH (100 ml) wasadded, and the mixture was stirred under bubbling of H₂ for 2 hours. Thecatalyst Pd/C was removed off by filtration through a pad of Celite. Thefiltrate was evaporated in vacuo to obtain a crude product (1.85 g),which was, in turn, subjected to silica gel chromatography (elutionsolvent, CHCl₃ :MeOH=5:1) to afford 1.74 g (91.1%) of the titledcompound.

¹ H-NMR spectrum (CD₃ OD) β ppm: 0.74 (3H, s, 18-CH₃), 0.9-2.9 (15H, m,estrogen CH and CH₂), 3.3-4.0 (7H, m, C₁₇ --H, and pyranose C₂ --H, C₃--H, C₄ H, C₅ --H and C₆ ₂), 4.6-4.8 (1H, m, pyranose C₁ --H), 6.52 (1H,s, Ar C₄ --H), and 7.11 (1H, s, Ar C₁ --H).

¹³ C-NMR spectrum (DMSO-d₆) δ ppm: 11.16, 22.76, 25.84, 26.93, 28.44,29.91, 36.63, 38.52, 42.75, 43.67, 49.52, 60.85, 69.89, 73.36, 75.96,77.10, 80.02, 103.38, 114.86, 115.57, 130.90, 143.31, and 144.71.

IR spectrum (KBr) cm⁻¹ : 3368, 2920, 2864, 1502, 1434, 1380, 1354, 1322,1292, 1264, 1204, 1068, and 884.

Silica gel TLC: Rf=0.21 (CHCl₃ :MeOH=5:1).

Solubility: 1.5 mg/0.5-1 ml (H₂ O).

Test Example 1

(Inhibitory effect of 2-hydroxyestradiol 17-glucoside on the lipidperoxidation of low-density lipoprotein)

Low-density lipoprotein (LDL) was prepared from hypercholesterolemicrabbit serum. 2-Hydroxyestradiol 17-(β-D-glucopyranoside) (2-OHE₂17-Glu) obtained according to the method of this invention (Example 4)was dissolved in distilled water and 2-hydroxyestradiol (2-OHE₂) as areference compound, in dimethylsulfoxide (DMSO), to give a finalconcentration of 500 μM. The reaction mixture contained the followingsin a final volume of 1 ml: 500 μg of LDL; 5 nmol of CuSO₄ ; and 5 nmolof either 2-OHE₂ 17-Glu or 2-OHE₂. As blank tests, distilled water orDMSO was added in place of 2-OHE₂ 17-Glu or 2-OHE₂. After incubation fora definite time at 30° C., the amount of lipid hydroperoxides generatedwas determined by the methylene blue method. Results are shown infollowing Table 1. It was found that 2-OHE₂ 17-Glu clearly inhibited theperoxidation of LDL and that its inhibitory potency was the same to thatof 2-OHE₂.

                  TABLE 1                                                         ______________________________________                                                   Incubation time                                                    Compounds    1 hr.  2 hr.     3 hr. 4 hr.                                     ______________________________________                                        2-OHE.sub.2 17-Glu                                                                         8.93   19.48     24.76 32.17                                     Water        35.41  80.36     123.79                                                                              167.72                                    2-OHE.sub.2  17.45  24.55     27.29 30.74                                     DMSO         35.51  74.35     115.77                                                                              150.07                                    ______________________________________                                    

Values in Table 1 indicate the amount of lipid peroxides (nmol/mgprotein).

Test Example 2

(Inhibitory effect of 2-hydroxyestradiol 2-glucoside on the lipidperoxidation of rat brain homogenate)

2-Hydroxyestradiol 2-(β-D-glucopyranoside) (2-OHE₂ 2-Glu) obtainedaccording to the method of this invention (Example 16) was dissolved inwater to give a concentration of 1 nM or 0.5 mM. The reaction mixturecontains the followings in a final volume of 2 ml: 1.98 ml of rat brainhomogenate (10 mg protein/ml) and 2-OHE₂ 2-Glu at a concentration of 5μM or 10 μM. After incubation at 37° C., lipid peroxide level wasdetermined by the thiobarbituric acid method.

Following Table 2 clearly shows that 2-OHE₂ 2-Glu strongly inhibited thelipid peroxidation in a concentration dependent manner.

                  TABLE 2                                                         ______________________________________                                                       Incubation time                                                Compounds        0 hr.  1 hr.    2 hr.                                                                              4 hr.                                   ______________________________________                                        2-OHE.sub.2 2-Glu (10 μM)                                                                   0.271  0.905    1.326                                                                              1.534                                   2-OHE.sub.2 2-Glu (5 μM)                                                                    0.271  1.124    1.867                                                                              3.167                                   Water            0.258  1.532    2.761                                                                              4.659                                   ______________________________________                                    

Values in Table 2 indicate the amount of lipid peroxides (nmol/mgprotein) in terms of malondialdehyde.

Test Example 3

(Inhibitory effect of 2-hydroxyestradiol 2-glucoside on the lipidperoxidation of rat liver homogenate)

A rat liver homogenate was prepared in 4 volume of 0.15M KCl. 2-OHE₂2-Glu was dissolved in distilled water and 2-OHE₂, in 1% DMSO, to give afinal concentration of 1 nM. The reaction mixture contained thefollowings in a final volume of 2 ml; 1.98 ml of rat liver homogenate(10 mg protein/ml) and 10 μM either 2-OHE₂ 2-Glu or 2-OHE₂. As blanktests, the same volume of distilled water or 1% DMSO was added in placeof 2-OHE₂ 2-Glu or 2-OHE₂. After incubation for 4 hours at 37° C., lipidperoxide level was determined by the thiobarbituric acid method. It wasfound that 2-OHE₂ 2-Glu and 2-OHE₂ inhibited the lipid peroxidation by77% and 73%, respectively.

Test Example 4

(Incorporation of glycosides of 2-hydroxyestradiol into red blood cells)

The reaction mixture contained 0.2 nmol of 2-OHE₂, 2-OHE₂ 17-Glu, or2-hydroxyestradiol 17-(β-maltoside) (2-OHE₂ 17-Mal, obtained in Example5) in 0.1 ml of 0.2% DMSO-0.1% ascorbic acid in phosphate bufferedsaline (PBS) and 0.9 ml of suspension of red blood cells in PBS. Afterincubation at 37° C. for several intervals, the mixture was centrifugedand supernatant fraction was obtained, and then the amount of eachglycoside of 2-hydroxyestradiol remained in the supernatant wasdetermined by HPLC. Following Table 3 shows that the incorporation of2-OHE₂ 17-Glu and 2-OHE₂ 17-Mal into red blood cells was markedlyretarded as compared with that of 2-OHE₂. This fact indicates thatglycosylated compounds of 2-OHE₂ are advantageous in a point of view asa long-acting drug.

                  TABLE 3                                                         ______________________________________                                        Incubation time                                                                         2-OHE.sub.2                                                                             2-OHE.sub.2 17-Glu                                                                        2-OHE.sub.2 17-Mal                            ______________________________________                                        0.5 min.  4.4       74.7        102                                           1.0 min.  4.4       54.1        90.1                                          2.0 min.  3.7       39.9        80.3                                          3.0 min.  --        20.6        86.9                                          4.0 min.  --        16.7        77.1                                          5.0 min.  --        13.5        81.0                                          ______________________________________                                    

Values in Table 3 indicate residual amount (%) in the incubation medium.

-: not determined.

Test Example 5

(Acute toxicity)

Male mice of ddY strain were received 2-OHE₂ 17-Glu or 2-OHE₂ 2-Glu byan intraperitoneal injection at doses of 250 mg, 500 mg, 750 mg, or 1000mg/kg body weight. All mice survived over an observation period of 2weeks and no abnormal symptom was found. Therefore, LD₅₀ of theglycosides was found to be over 1000 mg/kg body weight.

Medicine Preparation Example 1

(Drip infusion)

Ingredients listed below were blended in a conventional manner toprepare a drip infusion.

    ______________________________________                                        Prescription:                                                                 ______________________________________                                        Glycoside of catechol estrogen                                                                         50     mg                                            Ascorbic acid            5      mg                                            Isotonic sodium chloride solution                                                                      250    ml                                            ______________________________________                                    

Medicine Preparation Example 2

(Subcutaneous injection)

Ingredients listed below were blended in a conventional manner toprepare a subcutaneous injection.

    ______________________________________                                        Prescription:                                                                 ______________________________________                                        Glycoside of catechol estrogen                                                                         10    mg                                             Propylene glycol         0.8   ml                                             Distilled water for injection                                                                          1.2   ml                                             ______________________________________                                    

Medicine Preparation Example 3

(Tablet)

With use of following ingredients, a tablet was prepared in aconventional manner.

    ______________________________________                                        Prescription:                                                                 ______________________________________                                        Glycoside of catechol estrogen                                                                        50     (mg)                                           Lactose                 50                                                    Starch                  145                                                   Talc                    4                                                     Magnesium stearate      1                                                     ______________________________________                                    

What is claimed is:
 1. A glycoside of catechol estrogen having theformula of ##STR8## wherein X is carbonyl group or ##STR9## R₁₀ ishydroxyl group or glycosyloxy group, and R₂ is a hydrogen atom orethynyl group; R₁₁ is a hydrogen atom, hydroxyl group, or glycosyloxygroup; R₁₂ is hydroxyl group or glycosyloxy group; and R₁₃ is hydroxylgroup or glycosyloxy group,in which glycosyloxy group is selected fromthe group consisting of glucosyloxy, galactosyloxy, mannosyloxy,arabinosyloxy, ribosyloxy, xylosyloxy, fructosyloxy, rhamnosyloxy,fucosyloxy, maltosyloxy, cellobiosyloxy, lactosyloxy, maltotriosyloxy,maltotetraosyloxy, maltopentaosyloxy, maltohexaosyloxy,maltoheptaosyloxy, and sialosyloxy, and in this case, at least one ofR₁₀, R₁₁, R₁₂, and R₁₃ is glycosyloxy group as defined above.
 2. Acomposition comprising at least one of the glycosides of catecholestrogen of claim 1 in a lipid peroxidation suppressing amount and apharmaceutically acceptable carrier.